Abstract-Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114Ϯ1 to 133Ϯ1 mm Hg, PϽ0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2-transduced rats produced more 20-HETE (129Ϯ10 versus 97Ϯ7 pmol/mg protein, PϽ0.01) and less nitric oxide (NO; 4.2Ϯ1.6 versus 8.4Ϯ1 nmol nitriteϩnitrate/mg; PϽ0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation. T he endothelium plays a critical role in the short-and long-term regulation of the cardiovascular system. It serves as a protective barrier between tissues and circulating blood and functions to maintain vascular homeostasis by releasing bioactive factors in response to hemodynamic changes and blood borne signals. Such endothelial cell functions are impaired in many disease processes, including, diabetes, atherosclerosis, and hypertension.The expression of the cytochrome P450 (CYP) 4A and its catalytic activity as arachidonic acid -hydroxylase which produces 20-hydroxyeicosatetraenoic acid (20-HETE) has been linked to the regulation of vascular reactivity and tone and to the development of hypertension. This notion is substantiated by observations that 20-HETE promotes vasoconstriction, 1,2 that CYP4A expression and/or 20-HETE production is increased in vascular tissues of hypertensive animals, 3 and that interventions that decrease CYP4A expression and/or activity cause blood pressure to fall in animal models of hypertension. 4 -11 The vasoconstrictor action of 20-HETE is primarily attributed to inhibition of the large conductance Ca 2ϩ -activated K Their study demonstrated that 20-HETE attenuates acetylcholine-induced dilation in cremasteric arterioles of normotensive rats on a low salt diet and that inhibition of 20-HETE synthesis enhances arteriolar dilation to acetylcholine in hypertensive rats on a high salt diet. More recently, 20-HETE has been shown to functionally antagonize EDHF-mediated relaxation in small porcine coronary arteries. 20 In the pre...

OBJECTIVE: To transfect G156A mutant MGMT gene into peripheral blood mononuclear cells(PBMCs), peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells through recombinant adenovirus vector and detect the expression of ΔMGMT in these cells, to study the protective effect of the ΔMGMT gene for bone marrow cells against alkylating agent. METHODS: We obtained a full-length cDNA fragment encoding human O6-methylguanine-DNA-methytransferase (MGMT) gene from human liver tissue by RT-PCR. ΔMGMT cDNA was made by site-directed mutagenesis. Positive clones were identified by diagnostic restriction analysis and sequenced to confirm the presence of the desired mutation. ΔMGMT cDNA was released by digestion with XhoI and BamHI, purified and then ligated into adenovirus expression vector pAd5CMV-NpA. The recombinant adenovirus vectors pAd5CMV-ΔMGMT and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method. The recombination adenvirus were propagated, purified, and titrated to 1x1012pfu/mL. PBMCs, peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells were incubated with a mixture containing adenovirus. Fluorescence microscopy was employed to test the transfection of recombinant adenovirus vectors.The expression of ΔMGMT gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP. 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) and CFU-assays were used to detect the drug-resistance of the ΔMGMT transfected cells. RESULTS: MGMT gene was cloned successfully and the mutant G156A MGMT genes were obtained. High titer recombinant adenovirus had been made. Real time-PCR showed that mutant drug resistance gene expressed strongly. Western-Blot verified that cells infected with Adv-EGFP displayed EGFP immunoreactivity and basal levels of MGMT immunoreactivity; in contrast, cells infected with Adv-ΔMGMT showed a strong ΔMGMT immunoreactivity. Fluorescence microscopy showed EGFP positive in control group. The mutant drug resistance gene target cells conferred 12.4 ∼ 18.6-fold stronger resistances to BCNU and O6-benzylguanine (BG) in MTT test than control group. CFU-assays showed the colony-forming units was more detected in transfected group compared with vacant vector group (P<0.05). CONCLUSIONS: The results suggested that transduction of the mutant G156A MGMT into hematopoietic progenitor cells mediated by recombinant adenovirus vectors may lead to their selective resistance to the combined use of O6-benzylguanine and alkylating agents, and this study provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.